Role of Ionic Interactions in the Catalytic Mechanism of R67 Dihydrofolate Reductase

Author

Stephanie Nicole Hicks, University of Tennessee - Knoxville

Date of Award

12-2003

Degree Type

Thesis

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Liz Howell

Committee Members

Cynthia Peterson, Daniel Roberts, Frank Larimer

Abstract

The goal of this research is to investigate the role of K32 and K33 in the catalytic mechanism of R67 dihydrofolate reductase (DHFR). K32 is located in the active site pore and is the only charged residue in the active site while K33 is located on the surface of R67 DHFR. Both of the ligands for R67 DHFR, NADPH and dihydrofolate (DHF), have negative charges capable of forming ionic interactions with symmetry related K32 residues. NMR, DELPHI, and docking studies predict that K32 is involved in ionic interactions with the 2’phosphate of NADPH. Docking studies also predict that K32 participates in ionic interactions with DHF. Mutagenesis of K32 in the R67 DHFR homotetramer results in the formation of an inactive dimer. Thus, the role of K32 cannot directly be tested. Therefore, the role of ionic interactions in ligand binding and catalysis was experimentally examined using salt effects on K_{d1 (NADPH),} total heat of binding for folate (q_Total), K_{m (NADPH)}, K_{m (DHF)}, k_cat, k_cat/K_{m (NADPH)}, and k_cat/K_{m (DHF)}. Salt sensitivities of these parameters indicate that ionic interactions are involved in binding both cofactor and substrate. To examine the number of ionic contacts with each ligand, slopes of log-log plots of various binding and kinetic parameters vs. ionic strength can be used. However, one of the requirements that must be met for the slope to be directly proportional to the number of ionic contacts involved in binding and catalysis is that different anions produce the same effect on the slopes of these plots. Specific anion effects occur with R67 DHFR when salt sensitivities are compared in the presence of NaCl, NaF, or NaSCN. This makes quantation of the number of ionic contacts between R67 DHFR and each ligand difficult. In order to test the involvement of the 2’phosphate moiety of NADPH in ionic contacts, steady-state kinetics were performed using the alternate cofactor NADH. Significant effects were observed on k_cat/K_{m (NADH)} compared to k_cat/K_{m (NADPH)} revealing the 2' phosphate is most likely involved in an ionic interaction with R67 DHFR.

Direct analysis of the role of K32 in binding and catalysis was examined using a quadruplicated gene construct of R67 DHFR containing one to two asymmetric K32M mutations. One K32 mutation in each half pore of the enzyme results in a decrease in k_cat but has minimal effects on K_m values. On the other hand, two K32 mutations in the same half pore result in significant effects on the K_m values for NADPH and DHF as well as an enhancement of k_cat. The decrease in k_cat observed with the K32M double asymmetric mutants that possess a single K32M substitution in each half-pore support that R67 DHFR uses its symmetry to facilitate catalysis. This may arise from increasing the number of species available to form the transition state.

The increase in K_{m (NADPH)} and K_{m (DHF)} as well as the increase ink_cat observed with the K32M mutant that possesses two substitutions in the same half pore supports that an ionic interaction(s) is lost to reach the transition state. This mutant possesses 2 K32M substitutions in the same half pore (in the predicted binding site for DHF), resulting in a decrease in the affinity for DHF and NADPH in the ground state. These data suggest that K32 is involved in ionic contacts with DHF in the ground state. The unexpected elevation in K_{m (NADPH)} however_, may not reflect the true K_{d (NADPH)} for this mutant. Formation of the transition state is facilitated with this mutant. One possible scenario consistent with an increase in k_cat when two K32 residues are substituted in the same half pore involves the movement of DHF into a position that facilitates better overlap between the nicotinamide ring of NADPH and the pteridine ring of DHF thus facilitating the chemistry.

The salt sensitivities of the various binding and kinetic parameters, the large effects on k_cat/K_m in the presence of the alternate cofactor NADH, and the effects of the K32M double asymmetric mutants, reveal that K32 is most likely involved in at least one ionic contact with NADPH, and at least one ionic contact with DHF in the ground state. To allow formation of the transition state, one of these ionic interactions is lost.

Recommended Citation

Hicks, Stephanie Nicole, "Role of Ionic Interactions in the Catalytic Mechanism of R67 Dihydrofolate Reductase. " Master's Thesis, University of Tennessee, 2003.
https://trace.tennessee.edu/utk_gradthes/1977